NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that controls the transcription of DNA. NFκB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. It is now well accepted that the NF-κB pathway is involved in inflammatory diseases, cancer development and progression in human solid tumors.
In mammals, the NF-κB transcription factor family is composed of five members, RelA (p65), RelB, cRel (Rel), NF-κB1 (p50 and its precursor p105) and NF-κB2 (p52 and its precursor p100), and forms a collection of various homodimeric and heterodimeric complexes (Oeckinghaus A, et al, Cold Spring Harb Perspect Biol. 2009; Hayden M S and Ghosh S Cell. 2008).
The activity of the NF-κB subunit complexes is regulated by two major pathways. The first one, known as the classical or canonical NF-κB activation pathway, mainly applies to RelA:p50 dimers, which, under non-stimulated conditions, are sequestered in the cytoplasm through interactions with inhibitory proteins of the IκB family. Following stimulation with a broad range of stimuli such as TNF-α or IL-1, viruses, genotoxic agents and ionizing radiation, the IκB molecules are phosphorylated by the IκB kinase complex (IKK) at specific serine residues, leading to their ubiquitination and degradation by the proteasome pathway. RelA:p50 dimers are subsequently released and free to translocate to the nucleus where they activate transcription of various target genes (Ghosh, et al., Cell 2002). This pathway plays a major role in the control of innate immunity and inflammation (Baud, V. & Karin, M. Trends Cell Biol 2001; Bonizzi, G. & Karin, M. Trends Immunol 2004). The second pathway, the so-called alternative or non-canonical NF-κB signaling pathway, is stimulated by a more restricted set of cytokines that all belong to the TNF superfamily (e.g. BAFF, CD40L, LTβ). This pathway involves the upstream kinase NF-κB-inducing kinase (NIK) which activates IKKα, thereby leading to the phosphorylation and proteasome-dependent processing of p100, the main RelB inhibitor, resulting in RelB-p52 and RelB-p50 nuclear translocation and DNA binding (Derudder, E. et al. J Biol Chem 2003; Dejardin, E. et al. Immunity 2002; Xiao, G., et al, Mol Cell 2001; Coope, H. J. et al. Embo J. 2002; Claudio, E., et al. Nat Immunol 2002).
Most importantly, all studies point out to a crucial role for the RelB dependent alternative pathway in controlling the development, organization and function of secondary lymphoid organs and B-cell maturation and survival (Bonizzi, G. & Karin, M. Trends Immunol 2004; Dejardin, E. Biochem Pharmacol. 2006).
Beyond the alternative NF-κB signaling cascade, RelB-dependent DNA binding activity is negatively regulated at the nuclear level by several mechanisms, such as trapping in RelA/RelB or p100/RelB complexes, and specific serine phosphorylation (Marienfeld R, et al., J Biol Chem 2003; Jacque et al, PNAS 2005; Yilmaz Z B et al. Embo J 2003; Derudder E, et al. J Biol Chem 2003; Maier H J et al, J Biol Chem 2003). RelB containing dimers also display DNA binding specificity (Bonizzi G, et al. Embo J 2004; Fusco A J, et al., EMBO 2009; Natoli G and De Santa F, Cell Death Differ 2006), and RelB recruitment to some genes correlates with transcriptional down-regulation (IL12-p40), whereas in other cases (EBV-induced molecule 1 ligand chemokine (ELC) and macrophage-derived chemokine (MDC)), it increases transcriptional activity well over the level achieved by RelA or cRel (Saccani S, et al. Mol Cell 2003), further emphasizing the importance and unique role of RelB.
Several phosphorylation sites have been already characterized on the RelB protein. Phosphorylation at serine 368 has been shown to be required for NF-κB DNA binding activity, dimerization with other NF-κB subunits (p105/p50, p100/p52), and p100 half-life (Maier H J, et al., J Biol Chem 2003). Of note, no biological function has been associated to said phosphorylation and no inducer has been identified. Also, the threonine 84 and the serine 552 have been shown to undergo phosphorylation. This phosphorylation was found to be associated with the induction of RelB degradation by the proteasome in T cell lines (Marienfeld R, et al. Oncogene 2001). RelB is conserved through the mammal species and numerous homologs of the human RelB protein of SEQ ID NO:1 exist.
Activation of the canonical and non-canonical NFκB pathways has been involved in cell migration of a number of different cells.
For example, it has been shown that activation of the canonical NFκB pathway induces the expression of CXCR4 (Helbig et al, J. Biol. Chem. 2003) and the secretion of matrix metalloproteinases such as MMP9 (Sun et al, Carcinogenesis, 2012), so that it favors cancer cell migration and metastasis. Also, NFκB pathway activation has been shown to induce the secretion of MMP9 in macrophages (Rhee et al, Journal of Biochemistry and Molecular Biology, 2007) and to play a key role in regulating the immune response to infection. Finally, the NFκB pathway controls many genes involved in inflammation, and this pathway is found to be chronically active in many inflammatory diseases, such as inflammatory bowel disease, arthritis, sepsis, gastritis, asthma, atherosclerosis and others (Monaco et al, PNAS 2004). In view of all these implications, activation of the canonical NFκB pathway has been proposed to evaluate the clinical outcome of cancer patients, said activation being associated with a poor prognosis (Sun et al, Carcinogenesis, 2012). Also, inhibitors of the NF-κB pathway have been proposed to inhibit cancer cell migration, invasion, proliferation and tumor growth (Attoub et al, Journal of Medical Sciences, 2010) or inflammatory diseases (see anatabine, WO 2011/119722).
Interestingly, there are evidences indicating that the non-canonical NF-κB pathway and, in particular, the RelB subunit of NF-κB, is also involved in cell migration/invasion in a number of different cancer cells. This protein notably induces the more invasive mesenchymal phenotype in breast cancer cells (increase in snail, slug and vimentin protein expression along with a decrease in E-cadherin gene expression) (Wang et al, Nat. Cell. Biol. 2007). It has been also involved in invasion of gliomas (Lee, D. W., Et al., PLoS One) and prostate cancer (Guo, F., et al. Mol Immunol 2011).